Caffeoyl-CoA 3-O-methyltransferase genes

ABSTRACT

The present invention relates to new caffeoyl-CoA 3-O-methyltransferase genes isolated from plants and their use for the transformation of vectors, host organisms and plants and for the generation of plants which have an increased resistance to pests.

This application is a continuation of application Ser. No. 07/874,466,filed on Apr. 27, 1992 and now abandoned.

The present invention relates to new caffeoyl-CoA 3-O-methyltransferasegenes (called CCoAMT genes below) isolated from plants and to their usefor the transformation of vectors, host organisms and plants and for thegeneration of plants which have an increased resistance to pests.

The enzyme caffeoyl-CoA 3-O-methyltransferase, called CCoAMT below,catalyses the methylation of caffeoyl-CoA in a biosynthesis route, whichhas only recently been described, which leads from trans-4-coumaroyl-CoAto trans-feruloyl-CoA (Matern, U., and Kneusel, R. E. 1988,Phytoparasitica 16:153-170; Kneusel, R. E., Matern, U., and Nicolay, K.1989, Arch. Biochem. Biophys. 269:455 to 462; and Pakusch, A.-E.,Kneusel, R. E., and Matern, U., 1989, Arch. Biochem. Biophys. 271:488 to494).

Under fungal attack, plants reinforce their cell wall very rapidly byincorporation of cinnamic acids, followed by cross-linking thereof togive polymeric structures or build-up of lignin. Under these conditions,feruloyl-CoA is the preferred acyl donor both for the esterification ofcell wall polysaccharides and for lignification (reduction to coniferylalcohol). The speed and extent of the change in the cell wallessentially determine the course of the infection and the fate of theplants, "hypersensitive reaction" characterising complete resistance ofthe plants, associated with a particularly severe and rapid change inthe cell wall and the death of the cells directly affected. Thishypersensitive reaction is also observed in the resistance reaction ofplants to virus infections. It has only recently been discovered thatferuloyl-CoA is not formed in vivo in all cases by activation of ferulicacid, but is also formed by reaction of coumaroyl-CoA. Thecaffeoyl-CoA-specific methyltransferase which participates in thisreaction has scarcely any homology with previously known enzymes(Pakusch, A.-E., Matern, U., and Schiltz, E., 1991, Plant Physiol.95:137 to 143), is taxonomically widespread in plants and can be inducedtherein by, for example, fungal attack.

A large proportion of the world harvest of crop plants is constantlydestroyed by pests (in 1967 the loss of potential harvest was 35%;compare Chemistry of Pesticides, published by K. H. Buchel, John Wiley &Sons, New York, 1983, page 6). There is therefore an urgent need toresearch and utilise all possibilities which are capable of reducing orpreventing attack of crop plants by pests.

The new caffeoyl-CoA 3-O-methyltransferase genes, called CCoAMT genesbelow, have now been found, which can be incorporated into thehereditary factors (the genome) of plants which generate no CCoAMT oronly inadequate CCoAMT, whereby an increased resistance of these plantsto pests can be brought about.

It is surprising that it has been possible to find a new type ofresistance genes which can be incorporated as foreign or additional DNAinto the genome of plants, whereby an increased resistance of theresulting transgenic plants to pests is achieved. A particular advantageof the present invention is that--in contrast to, for example, the caseof increased accumulation of phytoalexins--it is not aimed at thegeneration of potentially toxic metabolites. There are therefore also notoxicological reservations, because the aim is the rapid synthesis inthe transformed plants of predominantly insoluble, antibioticallyinactive compounds which should function as physical barriers or preventpossible pathogen-induced, enzymatic lysis of cell wall polysaccharidesby acylation of the "substrate". In contrast to the transformation ofplants with genes of lytic enzymes, such as, for example, lysozyme oralso chitinase, which at best can become selectively active, theincreased readiness of plants to reinforce the cell wall offersprotection against every form of pathogens, including viruses. Thepresent invention here therefore follows a novel principle of plantprotection with wide application.

By CCoAMT genes, there are to be understood any nucleic acid (DNA)which, after its transcription into RNA and translation into protein,causes the formation of an enzyme which has the properties of a CCoAMT,this nucleic acid being isolated from its natural environment orintegrated into a vector or contained as "foreign" DNA or as"additional" DNA in a prokaryotic or eukaryotic DNA. By CCoAMT genesthere are also to be understood those CCoAMT genes which contain, attheir start and/or end, additional DNA sequences which do not or do notsubstantially impede the function of the genes. These DNA sequences,which are also called "gene units", are formed, for example, by excisionwith restriction enzymes, since no cleavage sites are available forcustomary restriction enzymes exactly at the start and at the end of thegene. The CCoAMT genes or the gene units can also carry at their endsDNA sequences which are appropriate for their handling (for example"linkers").

The CCoAMT genes (or the gene units) can exist in the form in which theyare contained in the genome of plants ("genomic" form, includingsequences which do not encode CCoAMT and/or do not have a regulatoryaction (such as introns)), or in a form which corresponds to the cDNA("copy" DNA) which is obtainable via mRNA with the aid of reversetranscriptase/polymerase (and no longer contains introns). The CCoAMTgenes can also be present in partially or completely synthetic form. Bysynthetic genes there are also understood those which are formed bynewly joining of parts of natural genes.

DNA segments or DNAs in the CCoAMT genes (or the gene units) accordingto the invention can be replaced by other DNA segments or DNAs whichhave essentially the same action.

In the present connection, by "foreign" DNA there is to be understoodDNA (in particular genes or gene units or componenents thereof) whichdoes not occur naturally in a certain prokaryotic or eukaryotic genome,but is taken up in this genome only as a result of intervention by man."Additional" DNA (in particular genes or gene units or componentsthereof) is intended to mean DNA which, although it occurs naturally inthe particular prokaryotic or eukaryotic genome, has been taken up inthis genome in an additional amount as a result of intervention by man.One or more copies of the "foreign" DNA or "additional" DNA can beincorporated, depending on requirements and on the nature of the case inquestion.

CCoAMT which is formed in plants or plant cells with the assistance ofthe CCoAMT genes (or the gene units) according to the invention meansany enzyme which acts like CCoAMT and, in plants, increases theirresistance to pests.

The preferred CCoAMT genes according to the invention are characterisedin that they hybridise with the CCoAMT-cDNA sequence contained in theplasmid pL2-4 or its components or with the cDNA sequence according toSEQ ID No: 1 or its components and encode CCoAMT.

CCoAMT genes which are preferred according to the invention are theCCoAMT genes which occur in parsley (Petroselinum crispum), carrots(Daucus carota), carnation (Dianthus caryophyllus) and safflower(Carthamus tinctorius), particularly preferably in parsley, and can beisolated from these.

The CCoAMT gene which is present (as a gene unit) in the form of thecDNA on the plasmid pL2-4 (which is described below in more detail) andthe DNA sequences which have essentially the same action are especiallypreferred as the CCoAMT gene according to the invention.

The cDNA contained on the plasmid was isolated from parsley. It consistsof a 5' untranslated leader sequence 370 nucleotides long and thecomplete protein-encoding region from position 371 to position 1093,followed by 67 nucleotides of a 3' untranslated sequence. The entirefragment was provided with EcoRI linkers on both sides and cloned intothe vector pGEM 7 (Promega Corp. Madison, Wis., USA). The residualsequence of the 3' untranslated region from position 1160 to 1258 is notpresent on the plasmid pL2-4. This poly-adenylation sequence can beprepared synthetically or replaced by another poly-A sequence. Thecomplete cDNA sequence can be seen from sequence protocol SEQ ID No:1.

The 5' untranslated region, the complete encoding region and 67nucleotides of the 3' untranslated region can be isolated in thecustomary manner with EcoRI on a fragment about 1170 long.

The chimaeric gene fusions of the TR promoter or the 35 S promoter withthe protein-encoding region of the CCoAMT genes, preferably of the genefrom parsley, in particular of the gene which corresponds to the cDNA onthe plasmid pL2-4, may be mentioned as particularly preferred. It hasbeen found that the CCoAMT genes which occur in plants have wide regionsof DNA sequence homology. On the basis of the sequence homology, theCCoAMT genes according to the invention can therefore be isolated fromplants in a simple manner with the aid of the cDNA contained on theplasmid pL2-4 or its components or the sequence information according toSEQ ID No: 1 in the customary manner using the known methods ofmolecular biology.

Possible plants from which CCoAMT genes according to the invention canbe isolated are practically all the monocotyledonous or dicotyledonousplants, preferably dicotyledonous plants, parsley, carrot, safflower andcarnation being mentioned by way of example and as preferred.

As already mentioned, the CCoAMT gene, or the encoding region thereof,which corresponds to the cDNA which lies on the plasmid pL2-4 ispreferred according to the invention. The gene or the coding region ofthe gene can be obtained in the customary manner with the aid of thecDNA.

The Escherichia coli strain DS pL2-4 contains the plasmid pL2-4. Thisstrain has been deposited at the Deutsche Sammlung von Mikroorganismen(DSM) German Collection of Microorganisms!, Mascheroder Weg 1b, D-3300Braunschweig, Federal Republic of Germany, in accordance with theconditions of the Budapest Treaty on the International Recognition ofDeposition of Microorganisms for the Purposes of Patent Proceedings(deposition date: 28th May 1991). It has been given deposition numberDSM 6536.

The present invention also relates to this strain and its mutants. Theplasmid pL2-4 deposited in this host can easily be obtained in therequired amounts in the customary manner by multiplication of the strainand subsequent isolation of the plasmid.

Functionally complete genes, such as the CCoAMT genes according to theinvention, consist of a component which has a regulatory action (inparticular a promoter) and the structural gene which codes for theprotein CCoAMT.

Both parts of the gene can be used independently of one another. It isthus possible to fuse the component having the regulatory action withanother DNA sequence (deviating from the CCoAMT gene) which is to beexpressed after incorporation into the plant genome. Since only a fewisolated promoters which can display their action in plants or plantcells are known, the promoters of the CCoAMT genes, to which the presentinvention likewise relates, are useful aids in the generation oftransformed plants or plant cells.

It is also possible to have the CCoAMT structural genes preceded by a"foreign" component having a regulatory action. This could beadvantageous if only specific regulatory active gene components (forexample those endogenous to the plant) can have a sufficient action incertain plants. The CCoAMT structural genes are therefore valuable unitswhich can be used independently and, as already mentioned, the presentinvention also relates to them. The CCoAMT genes according to theinvention can be separated into the components having a regulatoryaction and the structural genes by the customary methods. It is alsopossible to combine components of different naturally occurring CCoAMTgenes to give new functional "synthecic" genes. The complete naturallyoccurring CCoAMT genes (or the gene units) according to the inventionare preferably used. The CCoAMT structural gene which corresponds to thecDNA contained in the plasmid pL2-4 is preferred according to theinvention.

It is possible, with the aid of customary methods, to incorporate theCCoAMT genes (or the gene units) or their components in one or severalcopies (for example in tandem arrangement), preferably once, into anydesired prokaryotic (preferably bacterial) or eukaryotic (preferablyplant) DNA as "foreign" or "additional" DNA. Thus, for example, theprotein-encoding DNA corresponding to the cDNA can be provided withregulatory sequences and incorporated into plants. The present inventionrelates to the recombinant DNA "modified" in this way, which can beused, for example, for the transformation of plants or plant cells andis contained in the plants or plant cells after the transformation.

The CCoAMT genes (or the gene units) and/or their components and therecombinant DNA can be contained as "foreign" or "additional" DNA invectors (in particular plasmids, cosmids or phages), in transformedmicroorganisms (preferably bacteria, in particular Gram-negativebacteria, such as E. coli) and in transformed plant cells and plants orin the DNA thereof. The present invention relates to such vectors,transformed microorganisms (which can also contain these vectors) andthe transformed plant cells and plants and DNA thereof.

As already indicated, according to the invention the CCoAMT genes (orthe gene units) are incorporated in one or several copies (at the sameor different points of the genome) into the natural plant genome, italso being possible for different CCoAMT genes to be combined with oneanother. In the case of plants which already have the capacity forCCoAMT synthesis, the incorporation of one or more CCoAMT genesaccording to the invention can lead to considerably improved resistanceproperties. In the case of plants which contain no CCoAMT genes, anincreased resistance to pests is likewise achieved by incorporation ofsuch genes. If appropriate, only the structural genes according to theinvention are used, these being preceded by a regulatory DNA elementwhich may have been isolated from the particular plant.

The increased resistance of the transformed plant cells and plantsaccording to the invention is of importance for agriculture and forestryand for cultivation of ornamental plants, cultivation of medicinalplants and plant breeding. It is also advantageous in the culture ofplant cells, for example for the production of pharmaceutically usablesubstances, to have available plant cells which have increasedresistances to attack by microbial pests, in particular fungi.

The present invention thus also relates to a process for the preparationof transformed plant cells (including protoplasts) and plants (includingplant parts and seeds) having an increased resistance to pests, which ischaracterised in that

(a) one or more CCoAMT genes (or gene units) and/or components of theCCoAMT genes (or of the gene units) and/or recombinant DNA according tothe invention are inserted into the genome of plant cells (includingprotoplasts), and if appropriate

(b) complete transformed plants are regenerated from the transformedplant cells (including protoplasts) and if appropriate propagated, andif appropriate

(c) the desired plant parts (including seeds) are obtained from theresulting transformed plants of the parent generation or furthergenerations obtained therefrom.

Process steps (a), (b) and (c) can be carried out in the customarymanner by known processes and methods.

The present invention also relates to transformed plant cells (includingprotoplasts) and plants (including plant parts and seeds) which containone or more CCoAMT genes (or gene units) and/or components of the CCoAMTgenes (or of the gene units) as "foreign" or "additional" DNA, and tothose transformed plant cells and plants which are obtainable by theabove processes.

The present invention also relates to the:

(a) use of the CCoAMT genes (or of the gene units) and/or theircomponents and/or the recombinant DNA according to the invention and/orthe recombinant vectors according to the invention and/or thetransformed microorganisms according to the invention for thetransformation of plant cells (including protoplasts) and plants(including plant parts and seeds), the

(b) use of the transformed plant cells (including protoplasts) andplants (including plant parts and seeds) according to the invention forthe generation of propagation material and for the generation of newplants and propagation material thereof, the

(c) use of the CCoAMT genes according to the invention (or of the geneunits) and/or their components and/or the recombinant DNA according tothe invention for combating pests and the

d) use of the cDNA contained on the plasmid pL2-4 or its components andof the DNA sequences corresponding to the sequence information accordingto sequence protocol SEQ ID NO:1 for isolation of CCoAMT genes orcomponents thereof from plants and for the determination of CCoAMT genesin plants.

There are a number of different methods available for inserting theCCoAMT genes or the gene units or their components into the geneticmaterial of plants or plant cells as "foreign" or "additional" DNA. Thegene transfer can be carried out by the generally customary knownmethods, the expert being able to determine without difficulty theparticular method suitable.

The Ti plasmid from Agrobacterium tumefaciens is available as aparticularly favourable and widely applicable vector for the transfer offoreign DNA into genomes of dicotyledonous and monocotyledonous plants.The genetic material which encodes CCoAMT is inserted into the T-DNA ofsuitable Ti plasmids together with regulatory DNA sequences (for exampleZambryski et al. 1983) and transferred by infection of the plants,infection of plant parts or plant tissues, such as, for example, of leafdiscs, stems, hypocotyls, cotyledons, meristems and tissues issuingtherefrom, such as, for example, secondary embryos and calli, or bycoculture of protoplasts with Agrobacterium tumefaciens.

An alternative is the incubation of purified DNA which contains thedesired gene in plant protoplasts (for example Hain et al., 1985; Krenset al., 1982; Paszkowski et al., 1984) in the presence of polycations orcalcium salts and polyethylene glycol.

The DNA uptake can also additionally be promoted by an electric field(electroporation) (for example Fromm et al., 1986).

The DNA can also be introduced in a known manner via plant pollen, by"shooting" the pollen with physically accelerated particles which carrythe DNA (compare EP-A 0,270,356).

The plants are regenerated in a known manner with the aid of suitablenutrient media (for example Nagy and Maliga 1976).

In a preferred embodiment of the process according to the invention, thecDNA from the plasmid pL2-4 is cloned into an expression vector (forexample pRT101, Topfer et. al. 1988). The chimaeric constructed gene isthen isolated with the restriction enzyme Hind III and transferred in anintermediate vector (for example pCV001, Koncz and Schell 1986) toAgrobakterium tumefaciens (Koncz and Schell 1986).

Alternatively, the chimaeric constructed gene is cloned into the HindIII position of the plasmid PlGVneo 1103 (Hain et. al. 1985), and in aparticularly preferred embodiment the chimaeric constructed gene in theplasmid pLGVneo 1103 is transferred in the customary manner to plantprotoplasts by direct gene transfer (for example Hain et. al. 1985). Theplasmid can be in circular form, but is preferably in linear form here.

If this plasmid is used with a reporter gene, the kanamycin-resistantprotoplasts are then checked for expression of CCoAMT.

Transformed (transgenic) plants or plant cells are generated by theknown methods, for example by leaf disc transformation (for exampleHorsch et al. 1985) by coculture of regenerating plant protoplasts orcell cultures with Agrobacterium tumefaciens (for example Marton et al.1979, Hain et al. 1985) or by direct DNA transfection. Resultingtransformed plants are detected either by selection for expression ofthe reporter gene, for example by phosphorylation of kanamycin sulphatein vitro (Reiss et al. 1984; Schreier et al. 1985) or by the expressionof nopaline synthase (according to Aerts et al. 1983) or CCoAMT byNorthern blot analysis and Western blot analysis. The CCoAMT can also bedetected in a known manner with the aid of specific antibodies intransformed plants.

Culture of the transformed plant cells and regeneration to give completeplants are carried out by the generally customary methods with the aidof the particular suitable nutrient media.

Both the transformed plant cells and the transformed plants whichcontain the CCoAMT genes according to the invention (or the gene units)and to which the present invention relates exhibit a considerably higherresistance to pests, in particular phytopathogenic fungi.

In connection with the present invention, the term "plants" denotes bothcomplete plants and also parts of plants, such as leaves, seeds, tubers,cuttings and the like. "Plant cells" include protoplasts, cell lines,plant calli and the like. "Propagation material" denotes plants andplant cells which can be used for propagation of the transformed plantsand plant cells, and the present invention thus also relates to thismaterial.

In the present connection, the term "DNA sequences having essentiallythe same action" means that the invention also relates to thosemodifications in which the function of the CCoAMT genes and theircomponents is not impaired such that CCoAMT is no longer formed or theregulatory gene component is no longer active. Correspondingmodifications can be made by replacement, addition and/or removal of DNAsections, individual codons and/or individual nucleotides.

In the case of microorganisms which can be used according to theinvention, "mutants" denotes those modified microorganisms which stillhave the features essential for implementation of the invention, and inparticular contain the particular plasmids.

The plants which can be given resistance or an increased resistance topests by incorporation (transformation) of the CCoAMT genes according tothe invention (or the gene units) include practically all plants. Thereis of course a particular need for generating resistance in crop plants,such as forest plants, for example spruce, fir, Douglas fir, pine,latch, beech and oak, as well as plants which supply foodstuffs and rawmaterials, for example cereals (in particular wheat, rye, barley, oats,millet, rice and maize), potatoes, leguminous plants (such as pulses andin particular alfalfa and soybeans), vegetables (in particular cabbagevarieties and tomatoes), fruit (in particular apples, pears, cherries,grapes, citrus fruits, pineapples and bananas), oil palms, tea, cacaoand coffee shrubs, tobacco, sisal and cotton, and in medicinal plants,such as Rauwolfia and Digitalis. Potatoes, tomatoes and leguminousplants may be mentioned particularly preferably. The CCoAMT genesaccording to the invention are preferably incorporated into the genomeof plants as "foreign" DNA.

As pests against which resistances or increased resistances can beachieved with the aid of the CCoAMT genes according to the inventionthere may be mentioned animal pests, such as insects, mites andnematodes, as well as microbial pests, such as phytopathogenic fungi,bacteria and viruses. Microbial pests, in particular phytopathogenicfungi, are particularly singled out.

The harmful insects include, in particular, insects of the orders:

Orthoptera, Dermaptera, Isoptera, Thysanoptera, Heteroptera, Homoptera,Lepidoptera, Coleoptera, Hymenoptera and Diptera.

The harmful mites include, in particular:

Tarsonemus spp., Panonychus spp. and Tetranychus spp.

The harmful nematodes include, in particular:

Pratylenchus spp., Heterodera spp. and Meloidogyne spp.

The microbial pests include, in particular, the phytopathogenic fungi:

Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes,Ascomycetes, Basidiomycetes and Deuteromycetes.

The phytopathogenic bacteria include, in particular, thePseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceaeand Streptomycetaceae.

The virus diseases include, in particular, mosaic, dwarfing andyellowing viroses.

Some causative organisms of viral, fungal and bacterial diseases whichcome under the generic names listed above may be mentioned as examples,but not by way of limitation:

barley yellow dwarf virus (BYDV), potato virus Y (PVY), cucumber mosaicvirus (CMV), watermelon mosaic virus (WMV), Tristeza virus, tobaccomosaic virus (TMV), tobacco necrosis virus (TNV), beet necrotic yellowvein virus (BNYVV), rhizomania virus.

Xanthomonas species, such as, for example, Xanthomonas campestris pv.oryzae;

Pseudomonas species, such as, for example, Pseudomonas syringae pv.lachrymans;

Erwinia species, such as, for example, Erwinia amylovora;

Pythium species, such as, for example, Pythium ultimum;

Phytophthora species, such as, for example, Phytophthora infestans;

Pseudoperonospora species, such as, for example, Pseudoperonosporahumuli or Pseudoperonospora cubense;

Plasmopara species, such as, for example, Plasmopara viticola;

Peronospora species, such as, for example, Peronospora pisi or P.brassicae;

Erysiphe species, such as, for example, Erysiphe graminis;

Sphaerotheca species, such as, for example, Sphaerotheca fuliginea;

Podosphaera species, such as, for example, Podosphaera leucotricha;

Venturia species, such as, for example, Venturia inaequalis;

Pyrenophora species, such as, for example, Pyrenophora teres or P.graminea

(conidia form: Drechslera, syn: Helminthosporium);

Cochliobolus species, such as, for example, Cochliobolus sativus

(conidia form: Drechslera, syn: Helminthosporium);

Uromyces species, such as, for example, Uromyces appendiculatus;

Puccinia species, such as, for example, Puccinia recondita;

Tilletia species, such as, for example, Tilletia caries;

Ustilago species, such as, for example, Ustilago nuda or Ustilagoavenae;

Pellicularia species, such as, for example, Pellicularia sasakii;

Pyricularia species, such as, for example, Pyricularia oryzae;

Fusarium species, such as, for example, Fusarium culmorum;

Botrytis species, such as, for example, Botrytis cinerea;

Septoria species, such as, for example, Septoria nodorum;

Leptosphaeria species, such as, for example, Leptosphaeria nodorum;

Cercospora species, such as, for example, Cercospora canescens;

Alternaria species, such as, for example, Alternaria brassicae; and

Pseudocercosporella species, such as, for example, Pseudocerco sporellaherpotrichoides. Helminthosporium carbonum may furthermore be mentioned.

The present invention shall be illustrated in more detail with the aidof the following embodiment examples:

1. Isolation of the gene for CCoAMT from parsley

Plants and cell cultures from parsley (Petroselinum crispum) contain thegenes for CCoAMT which cause the formation of CCoAMT (size of theprotein 27,000 D; reaction with specific antiserum).

The known processes and methods of molecular biology such as aredescribed in detail, for example, in the following handbook were used inthe isolation of the CCoAMT genes: Maniatis, T., Fritsch, E. F.,Sambrook, J.: Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory, Second Edition 1989.

A "gene library" for parsley is first established: genomic DNA fromenriched cell nuclei (Bedbrook, J., Plant Molecular Biology Newsletter2, 24, 1981) is cut with the restriction enzyme NdeII such that DNAfragments having an average length of about 12,000 nucleotide pairs areformed. These fragments are cloned into the BamHI site of the lambdaphage EMBL4 (Frischauf et al., J. Mol. Biol. 170, 827-842, 1983), andthe phages are multiplied in E. coli. The phage population in itsentirety contains, cloned in sub fragments, the total genomic DNA ofparsley, and therefore also the genes for CCoMAT.

The genes for CCoMAT, their mRNA and the CCoMAT synthase cDNA eachcontain the same nucleic acid sequences, since they can be derived fromone another (gene→mRNA→cDNA). This means that the genes for CCoMAT canbe identified by specific hybridisation with CCoMAT-cDNA (compare SEQ IDNO:1) or with specific oligonucleotides which can be derived from thissequence. The phages with the genes are identified by hybridisation, andthen isolated and multiplied. The genomic DNA from parsley cloned inthis phage is mapped further by analysis with various restrictionenzymes, and the position of the CCoMAT genes is determined by furtherhybridisation experiments with cDNA sequences or syntheticoligonucleotides. Finally, the gene units are cut out of the phage bydigestion with restriction enzymes, cloned in the correspondingly cutplasmid vector and multiplied as recombinant plasmids.

Because of the sequence homologies, DNA sequences which correspond tothe sequences contained in the cDNA on the plasmid pL2-4 can be used asprobes for isolation of other CCoAMT genes according to the invention.

2. Transformation of tobacco

a) Culture of tobacco shoots and isolation of tobacco protoplasts

Nicotiana tabacum (Petit Havanna SR1) is propagated as a sterile shootculture on hormone-free LS medium (Linsmaier and Skoog 1965). Shootsections are transferred to fresh LS medium at intervals of about 6-8weeks. The shoot cultures are kept in 12 hours of light (1000-3000 lux)in a culture room at 24°-26° C.

For the isolation of leaf protoplasts, about 2 g of leaves (about 3-5 cmlong) are cut into small pieces (0.5 cm×1 cm) with a fresh razor blade.The leaf material is incubated in 20 ml of enzyme solution consisting ofK3 medium (Nagy and Maliga 1976), 0.4M sucrose, pH 5.6, 2% of ZellulaseR10 (Serva) and 0.5% of Macerozym R10 (Serra) at room temperature for14-16 hours. The protoplasts are then separated from cell residues byfiltration over a 0.30 mm and 0.1 mm steel sieve. The filtrate iscentrifuged at 100×g for 10 minutes. During this centrifugation, intactprotoplasts float and collect in a band at the top margin of the enzymesolution. The pellet of cell residues and the enzyme solution are suckedoff with a glass capillary. The prepurified protoplasts are made up to10 ml with fresh K3 medium (0.4M sucrose as an osmotic agent) andfloated again. The washing medium is sucked off and the protoplasts arediluted to 1-2×10⁵ /ml for culture or subsequent infection withAgrobacteria (coculture). The protoplast concentration is determined ina counting chamber.

b) Construction of a chimaeric CCoAMT gene and transfer intoAgrobacterium tumefaciens

The EcoRI fragment from pL2-4 (about 1.2 kb) is cloned into the EcoRIposition of the vector pRT 101 (Topfer et. al. 1988). This gives thecDNA the 35 S promoter of CaMV on its 5' end and a polyadenylationsequence from CaMV on its 3' end. This chimaeric constructed gene canthen be isolated functionally as a fragment of about 1.9 kb by cleavagewith HindIII. This HindIII fragment can then be transferred into anintermediate vector, for example pCV001 (Koncz and Schell, 1986) by thecustomary methods. Instead of the vectors mentioned, any other desiredexpression vectors and intermediate vectors which have correspondingcleavage sites can be employed, the expert easily being able to make asuitable choice on the basis of the above information. The resultingintermediate vector, which contains the CCoAMT gene, is transferred toAgrobacterium tumefaciens which contains a functional vir region (Konczand Schell 1986, van Haute et. al. 1983).

c) Transformation of regenerating tobacco protoplasts by coculture withAgrobacterium tumefaciens

The method of Marton et al. 1979 is used below, with minormodifications. The protoplasts are isolated as described and incubatedin a density of 1-2×10⁵ /ml in K3 medium (0.4M sucrose, 0.1 mg/l of NAA,0.2 ml in K3 medium (0.4M sucrose, 0.1 mg/l of NAA, 0.2 mg of kinetin)for 2 days in the dark and one to two days under weak light (500 lux) at26° C. As soon as the first divisions of the protoplasts occur, 30 μl ofan Agrobacterium suspension according to b) in minimal A (Am) medium(density about 10⁹ Agrobacteria/ml) are added to 3 ml of regeneratingprotoplasts. The duration of the coculture is 3-4 days at 20° C. in thedark. The tobacco cells are then introduced into 12 ml centrifuge tubes,diluted to 10 ml with seawater (600 mOsm/kg) and pelleted at 60×g for 10minutes. This washing operation is repeated a further 1-2 times in orderto remove the majority of the Agrobacteria. The cell suspension iscultured in a density of 5×10⁴ /ml in K3 medium (0.3M sucrose) with 1mg/l of NAA (naphthyl-1-acetic acid), 0.2 mg/l of kinetin and 500 mg/lof the cephalosporin antibiotic cefotaxime. The cell suspension isdiluted with fresh K3 medium every week and the osmotic value of themedium is reduced gradually by 0.05M sucrose (about 60 mOsm/kg) perweek. Selection with kanamycin (100 mg/l of kanamycin sulphate (Sigma),660 mg/g of active km) is started 2-3 weeks after the coculture in anagarose "bead type culture" (Shillito et al. 1983). Kanamycin-resistantcolonies can be distinguished from the background of retarded colonies3-4 weeks after the start of the selection.

d) Direct transformation of tobacco protoplasts with DNA. Calciumnitrate-PEG transformation

About 10⁶ protoplasms in 180 μl of K3 medium are carefully mixed in aPetri dish with 20 μl of aqueous DNA solution which contains 20 μg ofplasmid pCV001::CCoAMT (compare FIG. 3). The plasmid pCV001::CCoAMT isobtainable by known methods from the plasmid pCV001, pRT101 and pL2-4(compare FIGS. 1-3). 200 μl of fusion solution (0.1M calcium nitrate,0.45M mannitol, 25% of polyethylene glycol (PEG 6000), pH 9) are thencarefully added. After 15 minutes, 5 ml of washing solution (0.275Mcalcium nitrate pH 6) are added, and after a further 5 minutes theprotoplasts are transferred into a centrifuge tube and pelleted at 60×g.The pellet is taken up in a small amount of K3 medium and cultured asdescribed in the next section. Alternatively, the protoplasts can betransformed as described by Hain et al. 1985.

e) Culture of the protoplasts incubated with DNA and selection ofkanamycin-resistant calli

A modified "bead type culture" technique (Shillito et al. 1983) is usedfor the culture and selection of kanamycin-resistant colonies describedbelow. One week after treatment of the protoplasts with DNA (compare d),3 ml of the cell suspension are mixed with 3 ml of K3 medium (0.3Msucrose+hormones; 1.2% (Seaplaque) of LMT agarose (low melting agarose,Marine Colloids) in 5 cm Petri dishes. For this purpose, the agarose isautoclaved in the dry state and, after addition of K3 medium, is boiledup briefly in a microwave oven. After the agarose has solidified, theagarose discs ("beads") are transferred into 10 cm Petri dishes with theembedded tobacco microcalli for further culture and selection, and ineach case 10 ml of K3 medium (0.3M sucrose, 1 mg/l of NAA, 0.2 mg/l ofkinetin) and 100 mg/l of kanamycin sulphate (Sigma) are added. Theliquid medium is changed every week. During this procedure, the osmoticvalue of the medium is reduced in stages.

The replacement medium (K3+km) is reduced by 0.05M of sucrose (about 60mOsm) per week.

Timetable of the selection of kanamycin-resistant tobacco colonies afterDNA transformation:

    ______________________________________                                        0.4M   0.3M   0.25M    0.20M 0.15M  0.10M sucrose                                                                       in the                                                                        liquid                                                                        medium                              A E S                        K                                                1      2      3        4     5      6     weeks                                                                         after                                                                         DNA                                                                           Uptake                              ______________________________________                                         (K3 medium 1 mg of NAA, 0.2 mg of kinetin)                                    A = DNA uptake                                                                E = embedding in agarose                                                      S = selection with kanamycin (100 mg/l of kanamycin sulphate)                 K = kanamycinresistant colonies can be clearly distinguished from the         background                                                               

e) Regeneration of kanamycin-resistant plants

As soon as the kanamycin-resistant colonies have reached a diameter ofabout 0.5 cm, half of them are placed on regeneration medium (LS medium,2% of sucrose, 0.5 mg/l of benzylaminopurine BAP) and kept in theculture room in 12 hours of light (3000-5000 lux) at 24° C. The otherhalf are propagated as a callus culture on LS medium with 1 mg/l of NAA,0.2 mg/l of kinetin, 0.1 mg/l of BAP and 100 mg/l of kanamycin sulphate.When the regenerated shoots are about 1 cm in size, they are cut off andplaced on 1/2 LS medium (1% of sucrose, 0.8% of agar), without growthregulators, for rooting. The shoots are rooted on 1/2 MS medium with 100mg/l of kanamycin sulphate and later transferred into soil.

g) Transformation of leaf discs by Agrobacterium tumefaciens

For transformation of leaf discs (Horsch et al. 1985), leaves about 2-3cm long from sterile shoot cultures are stamped into discs of 1 cmdiameter and incubated with a suspension of appropriate Agrobacteria(about 10⁹ /ml) (compare c) in Am medium, see below) for about 5minutes. The infected pieces of leaf are kept on MS medium (see below)without hormones for 3-4 days at about 24° C. During this period,Agrobacterium grows over the pieces of leaf. The pieces of leaf are thenwashed in MS medium (0.5 mg/ml of BAP, 0.1 mg/ml of NAA) and placed onthe same medium (0.8% of agar) with 500 μg/ml of cefotaxime and 100μg/ml of kanamycin sulphate. The medium should be renewed after twoweeks. Transformed kanamycin-resistant shoots are visible after afurther 2-3 weeks.

Biochemical detection method of transformation

Neomycin phosphotransferase (NPT II) enzyme test

NPT II activity in plant tissue is detected as follows by in situphosphorylation of kanamycin as described by Reiβ et al. (1984) andmodified by Schreier et al. (1985). 50 mg of plant tissue arehomogenised on ice in 50 μl of extraction buffer (10% of glycerol, 5% of2-mercaptoethanol, 0.1% of SDS, 0.025% of bromophenol blue, 62.5 mM TrispH 6.8), with addition of glass powder, and centrifuged for 10 minutesin an Eppendorf centrifuge at 4° C. 50 μl of the supernatant are appliedto native polyacrylamide gel (145×110×1.2 mm; separating gel: 10% ofacrylamide, 0.33% of bisacrylamide, 0.375M Tris pH 8.8, collecting gel:5% of acrylamide, 0.165% of bisacrylamide, 0.125M Tris pH 6.8) andsubjected to electrophoresis overnight at 4° C. and 60 V. As soon as thebromophenol blue marker runs out of the gel, the gel is washed twicewith distilled water for 10 minutes and once for 30 minutes withreaction buffer (67 mM Tris-maleate, pH 7.1, 42 mM MgCl₂, 400 mMammonium chloride). The gel is placed on a glass plate of the same sizeand covered with a layer of 40 ml of 1% strength agarose in reactionbuffer which contains the substrates kanamycin sulphate (20 μg/ml) and20-200 μCi of ³² P ATP (Amersham). The sandwich gel is incubated for 30minutes at room temperature and a sheet of phosphocellulose paper P81(Whatman) is then laid over the agarose. Four layers of 3 MM filterpaper, (Whatman) and a few paper handkerchiefs are stacked on top. Thetransfer of radioactive kanamycin phosphate phosphorylated in situ ontothe P81 paper is stopped after 3-4 hours. The P81 paper is incubated for30 minutes in a solution of proteinase K and 1% of sodium dodecylsulphate (SDS) at 60° C. and then washed 3-4 times in 250 ml of 10 mMphosphate buffer pH 7.5 at 80° C., dried and autoradiographed for 1-12hours at -70° C. (XAR5 film from Kodak).

4. Transformation of Solanum tuberosum (potato)

The transformation was carried out in exactly the manner described inEP-A-0,242,246, pages 14 to 15, the Agrobacteria containing Ti plasmidswhich carry the CCoAMT gene or the CCoAMT genes.

All the percentage data in the above examples relate to percentages byweight, unless stated otherwise.

The presence of the CCoAMT genes in the plant cells and plants (tobacco)obtained according to the above examples was confirmed by Southern blotanalysis. The expression of the CCoAMT genes was detected by Northernblot analysis, and CCoAMT was detected with the aid of specificantibodies.

Some of the media employed in the transformation of plants and plantcells are described below:

    ______________________________________                                        Am medium                                                                     3.5 g of           K.sub.2 HPO.sub.4                                          1.5 g of           KH.sub.2 PO.sub.4                                          0.5 g of           Na.sub.3 citrate                                           0.1 g of           MgSO.sub.4 × 7H.sub.2 O                                1 g of           (NH.sub.4).sub.2 SO.sub.4                                    2 g of           glucose to 1 l                                             Medium for sterile shoot culture of tobacco                                   Macroelements  1/2 of the concentration of the MS salts                       Microelements  1/2 of the concentration of the MS salts                       Fe-EDTA        Murashige and Skoog (MS)                                       Myo-inositol           100    mg/l                                            Sucrose                10     mg/l                                            Agar                   8      g/l                                             Vitamins                                                                      Ca panthotenate        1      mg/l                                            Biotin                 10     mg/l                                            Nicotinic acid         1      mg/l                                            Pyridoxine             1      mg/l                                            Thiamine               1      mg/l                                            pH 5.7 before autoclaving                                                     K3 medium                                                                     For culture of Nicotiana tabacum petit Havana SR1,                            Nicotiana tabacum Wisconsin 38 and Nicotiana plumagini-                       folia protoplasts (Nagy and Maliga, 1976)                                     Macroelements                                                                              NH.sub.4 NO.sub.3                                                                           250    mg/l                                                     KNO.sub.3     2500   mg/l                                                     CaCl.sub.2.2H.sub.2 O                                                                       900    mg/l                                                     MgSO.sub.4.7H.sub.2 O                                                                       250    mg/l                                                     NaH.sub.2 PO.sub.4.1H.sub.2 O                                                               150    mg/l                                                     (NH.sub.4).sub.2 SO.sub.4                                                                   134    mg/l                                                     CaHPO.sub.4.1H.sub.2 O                                                                      50     mg/l                                        Microelements                                                                              H.sub.3 BO.sub.3                                                                            3      mg/l                                                     MnSO.sub.4.1H.sub.2 O                                                                       10     mg/l                                                     ZnSO.sub.4.4H.sub.2 O                                                                       2      mg/l                                                     KI            0.75   mg/l                                                     Na.sub.2 MoO.sub.4.2H.sub.2 O                                                               0.25   mg/l                                                     CuSO.sub.4.5H.sub.2 O                                                                       0.025  mg/l                                                     CoCl.sub.2.6H.sub.2 O                                                                       0.025  mg/l                                        Fe-EDTA      Na.sub.2 EDTA 37.2   mg/l                                                     FeSO.sub.4.7H.sub.2 O                                                                       27.8   mg/l                                        Inositol                   100    mg/l                                        Sucrose                    137    g/l                                                                           (= 0.4M)                                    Xylose                     250    mg/l                                        Vitamins     Nicotinic acid                                                                              1      mg/l                                                     Pyridoxine    1      mg/l                                                     Thiamine      10     mg/l                                        Hormones     NAA           1.0    mg/l                                                     Kinetin       0.2    mg/l                                        pH 5.6                                                                        Sterilise filter                                                              Linsmaier and Skoop medium (Linsmaier and Skoog 1965)                         For culture of regenerating protoplasts and for tissue                        culture of tobacco tumours and callus. Linsmaier and                          Skoog (LS) medium is Murashige and Skoog medium                               (Murashige and Skoog, 1962) with the following modifications:                 thiamine is weighed in at a higher concentration of                           0.4 mg/l instead of 0.1 mg/l;                                                 glycine, pyridoxine and nicotinic acid are absent.                            Macroelements                                                                              NH.sub.4 NO.sub.3                                                                           1650   mg/l                                                     KNO.sub.3     1900   mg/l                                                     CaCl.sub.2.2H.sub.2 O                                                                       440    mg/l                                                     MgSO.sub.4.7H.sub.2 O                                                                       370    mg/l                                                     KH.sub.2 PO.sub.4                                                                           170    mg/l                                        Microelements                                                                              H.sub.3 BO.sub.3                                                                            6.2    mg/l                                                     MnSO.sub.4.1H.sub.2 O                                                                       22.3   mg/l                                                     ZnSO.sub.4.4H.sub.2 O                                                                       8.6    mg/l                                                     KI            0.83   mg/l                                                     Na.sub.2 MoO.sub.4.2H.sub.2 O                                                               0.25   mg/l                                                     CuSO.sub.4.5H.sub.2 O                                                                       0.025  mg/l                                                     CoCl.sub.2.6H.sub.2 O                                                                       0.025  mg/l                                        Fe-EDTA      Na.sub.2 EDTA 37.2   mg/l                                                     FeSO.sub.4.7H.sub.2 O                                                                       27.8   mg/l                                        Inositol                   100    mg/l                                        Sucrose                    30     g/l                                         Agar                       8      g/l                                         Vitamins     Thiamine      0.4    mg/l                                        Hormones:    NAA           1      mg/l                                                     Kinetin       0.2    mg/l                                        pH 5.7 before autoclaving                                                     ______________________________________                                    

The following literature can be cited for transformation of plants andplant cells:

Fraley R. T., Rogers S. G., Horsch R. B., Sanders P. R., Flick J. S.,Adams S. P., Bittner M. L., Brand L. A., Fink C. L., Fry J. S., FallupiG. R., Goldberg S. B., Hoffmann N. L., Woo S. C. (1983). Expression ofbacterial genes in plant cells. Proc. Natl. Acad. Sci. USA 80:4803-4807.

Fromm M E, Taylor L P, Walbot V (1986) Stable transformation of maizeafter gene transfer by electropotation. Nature 319:791-793

Hain, R., Stabel, P., Czernilofsky, A. P., Steinbiβ, H. H.,Herrera-Estrella, L., Schell, J. (1985) Uptake, integration, expressionand genetic transmission of a selectable chimeric gene by plantprotoplasts. Molec Gen Genet 199: 161-168.

Hernalsteens J P, Thia-Tong L, Schell J, Van Montagu M (1984) AnAgrobacterium-transformed Cell culture from the monocot Asparagusofficinalis. EMBO J 3:3039-3041

Herrera-Estrella L., De Block M., Messens E., Hernalsteens J P., vanMontagu M., Schell J. (1983) EMBO J. 2: 987-995.

Horsch R B, Fry J E, Hoffmann N L, Eichholtz D, Rogers S G, Fraley R T(1985) A simple and general method for transferring genes into plants.Science 277:1229-1231

Krens F H, Molendijk L, Wullems G J, Schilperoort R A (1982) in vitrotransformation of plant protoplasts with Ti-plasmid DNA. Nature296:72-74

Koncz C, Schell J (1986) The promotor of T_(L) -DNA gene 5 controls thetissue-specific expression of chimaeric genes carried by a noval type ofAgrobacterium linary vector. Mol. Gen. Genet. (1986) 204: 338-396Linsmaier D M, Skoog F (1965) Organic growth factor requirements oftobacco tissue cultures. Physiol plant 18: 100-127

Marton L, Wullems G J, Molendijk L, Schilperoort P R (1979) In vitrotransformation of cultured cells from Nicotiana tabacum by Agrobacteriumtumefaciens. Nature 277: 1229-131

Nagy J I, Maliga P (1976) Callus induction and plant regeneration frommesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol78:453-455

Paszkowski J, Shillito R D, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I(1984) Direct gene transfer to plants. EMBO J 3: 2717-2722

Shillito R D, Paszkowski J. Potrykus I (1983) Agarose plating and Beadtype culture technique enable and stimulate development ofprotoplast-derived colonies in an number of plant species. Pl Cell Rep2: 244-247 Van den Elzen P J M, Townsend J, Lee K Y, Bedbrook J R (1985)A chimaeric resistance gen as a selectable marker in plant cells. PlantMol. Biol. 5, 299-302.

Van den Elzen P J M, Townsend J, Lee K Y, Bedbrook J R (1985) Achimaeric resistance gen as a selectable marker in plant cells. PlantMol. Biol. 5, 299-302.

Velten J, Velten L, Hain R, Schell J (1984) Isolation of a dual plantpromotor fragment from the Ti Plasmid of Agrobacterium tumefaciens. EMBOJ 12: 2723-2730

Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983)Intergenic transfer and excharge recombination of restriction fragmentsclones in pBR322: a novel strategy for the reversed genetics of Tiplasmids of /Agrobacterium tumefacines. EMBO J 2: 411-418.

Zambryski P, Joos H, Genetello C, van Montagu M, Schell J (1983)Ti-plasmid vector for the introduction of DNA into plant cells withoutaltering their normal regeneration capacity, EMBO J 12: 2143-2150.

Reiss, B., Sprengel, Will H., and Schaller H (1984) A new sensitivemethod for qualitative and quantitative assay of neomycinphosphotransferase in crude cell tracts, GENE 1081: 211-217

Schreier P. H., Seftor E. A., Schell J. and Bohnert H. J. (1985) The useof nuclear-encoded sequences to direct the light-regulated synthesis andtransport of a foreingn protein into plant chloroplasts, EMBO J Vol. 4,No. 1: 25-32.

The following published patent applications may furthermore bementioned:

    ______________________________________                                        EP-A 116,718        EP-A-126,546                                              EP-A 159,418        EP-A-164,597                                              EP-A 120,515        EP-A-175,966                                              EP-A-120,516        WO 84/02913                                               EP-A-172,112        WO 84/02919                                               EP-A-140,556        WO 84/02920                                               EP-A-174,166        WO 83/01176                                               EP-A-122,791                                                                  ______________________________________                                    

The increased resistance of the transformed plants according to theinvention may be illustrated with the aid of the following example:

Detection of the increased resistance of transformed plants

EXAMPLE A

To test for an increased resistance to plant diseases, the plants areinoculated with a pathogen and the degree of attack is used asparameter. Botrytis cinerea Pers. is used as the test pathogen.

The tobacco plants are pregrown in tissue culture and subsequentlypotted in standard soil (Balster) in pots (d=11 cm) in a greenhouse andgrown in the greenhouse at 23° C. and 70-80% relative atmospherichumidity until the start of the experiment. The plants are supplied withwater and fertiliser as required. For inoculation, the leaves of theplants (3-4 weeks after transfer into the greenhouse) are sprayed with aspore suspension of the pathogen until dripping wet. The plants are thenincubated at 100% relative atmospheric humidity and 10°-20° C. After 4-8days, the state of health of the plants is determined in percent withthe aid of the leaf area attacked.

The transformed tobacco plants into which a CCoAMT gene according to theinvention had been inserted exhibit a significantly lower attack by B.cinerea than the of the non-transformed plants.

Explanations on diagrams 1 to 3 (FIG. 1 to FIG. 3)

Abbreviations used

    ______________________________________                                        1              Start of the encoding region                                   2              End of the encoding region                                     CaMV           Cauliflower mosaic virus                                       Cb.sup.R       Carbenicillin resistance gene                                  E              EcoRI cleavage site                                            H              HindIII cleavage site                                          Km.sup.R       Kanamycin resistance gene for                                                 plants                                                         P35S           CaMV35S promoter                                               pA35S          Polyadenylation sequence of CaMV                               RV             EcoRV                                                          S              SST1 cleavage site                                             Arrow direction                                                                              Direction of the promoter and of                                              the gene                                                       LB             Left border sequence of the T-DNA                                             of A. tumefaciens                                              RB             Right border sequence of the T-DNA                                            of A. tumefaciens                                              ______________________________________                                    

FIG. 1: FIG. 1 represents a diagram of the plasmid pL2-4 which containsthe protein-encoding sequence of the CCoAMT gene (compare also SEQ IDNO:1) on the EcoRI fragment.

FIG. 2: FIG. 2 represents a diagram of the plasmid pRT101::CCoAMT whichcontains a chimaeric CCoAMT gene.

FIG. 3: FIG. 3 represents a diagram of the plasmid pCV001::CCoAMT whichcontains a chimaeric CCoAMT gene.

Preferred hybridisation conditions

As mentioned above, the preferred CCoAMT genes according to theinvention are characterised in that they hybridise with the CCoAMT-cDNAsequence contained in the plasmid pL2-4 or its components or with thecDNA sequence according to SEQ ID No: 1 or its components and encodeCCoAMT.

Preferably moderate stringency conditions are used. Moderate stringencyconditions means preferably at 58° to 65° C. (particularly preferred at63° C.) in 3 to 4 times concentrated SSC.

If this method is used to isolate CCoAMT genes from other sources,normally a population of cDNA's with related sequences are obtainedwhich can e. g. be expressed in E. coli. The enzyme activity can bedetermined (e. g. according to Pakusch et al, Arch. Biochem. Biophys.271 (1989), pp. 488-494) and the desired cDNA can be isolated.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1258 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: parsley                                                         (H) CELL LINE: parsley cell culture;                                          Petroselinum crispum                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mature peptide                                                  (B) LOCATION: 371 to 1093                                                     (D) OTHER INFORMATION: codes for caffeoyl-CoA                                 3-O- methyltransferase                                                        from parsley                                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: untranslated region                                             (B) LOCATION: 1 to 370; 1094 to 1258                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGAGCTCAGGCAGATGCACTTAATCAGCTAACCACTGATG40                                    ACCTTGAAGGACAGTTTGCATTGCTGGAGACTTCATCAGT80                                    CGATGATGATCTTGCGAGTTTGAAGAAAGAATTGTCTGGA120                                   AGTAGAAAGAAAGGACAGCTTCCGCCAGGAAGAACTACTG160                                   CTGCCTCAAACTCGGGATTTCCATTCAGAGAAACTGAAAT200                                   TGAGAATGAGCTAAACGAACTTAGGAGAAAAGCCGCTGAT240                                   TACTAAATATACAACTCTGCATATGTTCACTATGACTGCA280                                   CCTACTGCATCTACAAATGTACTTTTTGGTTGATTGTGGA320                                   CATTCTATACATACGTTAAGAGGCAGATTTGTCGTTTGGA360                                   CAAATTCCAGATGGCTTCTAATGGTGAATCTAAACATTCA400                                   MetAlaSerAsnGlyGluSerLysHisSer                                                GAAGTTGGGCACAAGAGTCTTTTGCAGAGTGATGCTCTT439                                    GluValGlyHisLysSerLeuLeuGlnSerAspAlaLeu                                       TATCAGTATATACTTGAAACAAGTGTGTACCCAAGAGAA478                                    TyrGlnTyrIleLeuGluThrSerValTyrProArgGlu                                       CCAGAGGCAATGAAAGAGCTTAGAGAAGTCACCGCAAAG517                                    ProGluAlaMetLysGluLeuArgGluValThrAlaLys                                       CATCCATGGAATCTGATGACAACATCAGCTGATGAAGGG556                                    HisProTrpAsnLeuMetThrThrSerAlaAspGluGly                                       CAGTTCTTGAACATGCTTTTGAAGCTCATCAATGCCAAA595                                    GlnPheLeuAsnMetLeuLeuLysLeuIleAsnAlaLys                                       AACACCATGGAGATTGGTGTTTACACTGGTTATTCTCTC634                                    AsnThrMetGluIleGlyValTyrThrGlyTyrSerLeu                                       CTTGCCACTGCCCTGGCTCTTCCAGATGATGGAAAGATT673                                    LeuAlaThrAlaLeuAlaLeuProAspAspGlyLysIle                                       TTGGCAATGGATATCAACAGAGAAAACTATGAAATTGGA712                                    LeuAlaMetAspIleAsnArgGluAsnTyrGluIleGly                                       TTACCCATCATTGAAAAAGCTGGAGTTGGTCACAAAATT751                                    LeuProIleIleGluLysAlaGlyValGlyHisLysIle                                       GACTTCAGAGAAGGCCCAGCTTTGCCTGTTCTTGATCAT790                                    AspPheArgGluGlyProAlaLeuProValLeuAspHis                                       ATGCTTGAAGATGGAAAGTATCATGGAACATTTGATTTT829                                    MetLeuGluAspGlyLysTyrHisGlyThrPheAspPhe                                       GTATTTGTTGATGCTGACAAGGATAACTATATCAACTAC868                                    ValPheValAspAlaAspLysAspAsnTyrIleAsnTyr                                       CACAAGAGATTAATTGATTTAGTAAAAATCGGAGGACTT907                                    HisLysArgLeuIleAspLeuValLysIleGlyGlyLeu                                       ATCGGCTACGACAACACCCTATGGAATGGTTCTGTGGCT946                                    IleGlyTyrAspAsnThrLeuTrpAsnGlySerValAla                                       CAGCCAGCTGATGCTCCAATGAGAAAGTATGTAAGGTAC985                                    GlnProAlaAspAlaProMetArgLysTyrValArgTyr                                       TACAGAGACTTTGTGGATTGAGCTTAACAAGCTCTGGCC1024                                   TyrArgAspPheValIleGluLeuAsnLysAlaLeuAla                                       GCTGATCCCAGGATTGAGATCTGTATGCTTCCTGTTGGT1063                                   AlaAspProArgIleGluIleCysMetLeuProValGly                                       GATGGAGTTACCCTGTGCCGTCGTATCAGCTGATTATCTA1103                                  AspGlyValThrLeuCysArgArgIleSer                                                ACTGAAATTTGAGATATTATTTCACAATGTTTTAAGAAAT1143                                  GGAATACTTTTGCTTTGATTGTATCTTCCTATGTTTCTTG1183                                  TTGAATTTGCAATGTGCATTATTGATGATGAATATATTCA1223                                  TAATTGATGTTGAAAAAAAAAAAAAAAAAAAAAAA1258                                       __________________________________________________________________________

We claim:
 1. An isolated and purified DNA sequence comprising anucleotide sequence that encodes the caffeoyl-CoA3-O-methyltransferaseencoded by the nucleotide sequence of SEQ ID NO:
 1. 2. An isolated andpurified DNA sequence according to claim 1 that consists of nucleotidesequence 371 to 1093 of SEQ ID No:
 1. 3. An isolated and purified DNAsequence consisting of SEQ ID No:
 1. 4. A chimeric gene comprising:(a) apromoter selected from the group consisting of the TR promoter and the35S promoter;operably linked to: (b) a DNA sequence comprising anucleotide sequence that encodes the caffeoyl-CoA3-O-methyltransferaseencoded by the nucleotide sequence of SEQ ID NO:
 1. 5. A chimeric geneaccording to claim 4, wherein the promoter is the TR promoter and thenucleotide sequence encoding caffeoyl-CoA3-O-methyltransferase comprisesthe nucleotide sequence 371 to 1093 of SEQ ID No:
 1. 6. A chimetic geneaccording to claim 5, wherein the nucleotide sequence encodingcaffeoyl-CoA3-O-methyltransferase consists of the nucleotide sequence371 to 1093 of SEQ ID No:
 1. 7. A chimeric gene according to claim 4,wherein the promoter is the 35S promoter and the nucleotide sequenceencoding caffeoyl-CoA3-O-methyltransferase comprises the nucleotidesequence 371 to 1093 of SEQ ID NO:
 1. 8. A chimeric gene according toclaim 7, wherein the nucleotide sequence encodingcaffeoyl-CoA3-O-methyltransferase consists of the nucleotide sequence371 to 1093 of SEQ ID No:
 1. 9. A vector containing DNA according toclaim
 1. 10. A vector containing DNA according to claim
 3. 11. A vectorcontaining DNA according to claim
 4. 12. A vector containing DNAaccording to claim
 5. 13. A vector containing DNA according to claim 6.14. A vector containing DNA according to claim
 7. 15. A vectorcontaining DNA according to claim
 8. 16. A microorganism transformedwith a vector according to claim
 9. 17. A microorganism transformed witha vector according to claim
 10. 18. A microorganism transformed with avector according to claim
 11. 19. A microorganism transformed with avector according to claim
 12. 20. A microorganism transformed with avector according to claim
 13. 21. A microorganism transformed with avector according to claim
 14. 22. A microorganism transformed with avector according to claim
 15. 23. Escherichia coli strain Escherichiacoli DS pL2-4.